Biomimetic organic/inorganic composites, processes for their production, and methods of use

ABSTRACT

The subject invention concerns a composite comprising an organic fluid-swellable, fibrous matrix, such as collagen, and a mineral phase, such as calcium carbonate or phosphate mineral phase, for use as a biomimetic of bone. In another aspect, the subject invention concerns a process for making a composite involving the inclusion of acidic polymers to a supersaturated mineralizing solution, in order to induce an amorphous liquid-phase precursor to the inorganic mineral, which is then absorbed (pulled by capillary action) into the organic matrix. Advantageously, once solidified, a high mineral content can be achieved, with the inorganic mineral crystals embedded within the collagen fibers (intrafibrillarly) and oriented such that they are aligned along the long axes of the fibers of the organic matrix, thereby closely mimicking the natural structure of bone. The present invention further concerns a method of treating a patient suffering from a bone defect by applying a biomimetic composite to the bone defect site.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.10/691,002, filed Oct. 22, 2003, which is a continuation-in-part of U.S.application Ser. No. 10/418,843, filed Apr. 18, 2003, which claims thebenefit of U.S. Provisional Application No. 60/373,801, filed Apr. 18,2002, which are hereby incorporated by reference in their entirety,including all figures, tables, and drawings.

GOVERNMENT SUPPORT

The subject invention was made with government support under a researchproject supported by National Science Foundation Grant No. ECS-9986333.The government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Synthetic bone graft material made to closely resemble natural bonewould be a useful replacement for natural bone. Acceptable syntheticbone can avoid the problem of availability and harvesting of autogenousbone and the risks and complications associated with allograft bone,such as risks of infection, disease, and viral transmission.

Natural bone is a composite material consisting of both water andorganic and inorganic solid phases. Bone has a hard structure becauseits organic extracellular collagenous matrix is impregnated withinorganic crystals, principally hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂).Calcium and phosphate account for roughly 65% to 70% of the bone's dryweight. Collagen fibers compose approximately 95% of the extracellularmatrix and account for 25% to 30% of the dry weight of bone. The organicmaterial gives bone its flexibility and resilience, while the inorganicmaterial gives bone its strength and rigidity (modulus), and theorganization of the two phases provides a high degree of toughness tothe composite. A thorough review of bone structure from the angstromlevel (mineral crystal) to the micron level (lamellae) has beenpresented (Weiner, S. et al. [1992] FASEB, 6:879-885).

Surrounding the mineralized collagen fibers is a ground substanceconsisting of protein-polysaccharides, or glycosaminoglycans, primarilyin the form of proteoglycan macromolecules. The glycosaminoglycans serveto cement together the various layers of mineralized collagen fibers.The individual collagen molecules self-assemble to form triple helices,which assemble into collagen fibrils, which then assemble intomicroscopic fibers. Within the packing of the collagen fibrils/fibersare distinct gaps, sometimes called hole zones. These hole zones arecreated by the staggered arrangement of tropocollagen molecules (triplehelical rods), which leads to periodicity of the hole and overlap zones.Various models have been proposed where these hole zones are completelyisolated from each other, or are contiguous and together form a groove.Within these hole zones, mineral crystals form. The mineral crystals infinal form nucleate and grow within the fibrils (intrafibrillarmineralization), as well as into the interstitial spaces (interfibrillarmineralization) (Landis, W. J. et al. [1993] J. Struc. Biol. 110:39-54).The mineral crystals in final form are a carbonated apatite mineral(dahllite), but initially may form as an amorphous calcium phosphatephase, which then transforms into the apatite (or possibly via anoctacalcium phosphate precursor, which naturally forms plates). Theapatite platelets of bone are of nanoscopic dimensions (only a few unitcells thick), and are densely packed into the type I collagen fibrilsdue to the intrafibrillar mineralization mechanism, and are welloriented with their c-axis (in the [001] direction) parallel to the longaxis of the collagen fibrils. Because of the nature of the packing, theorientation of the collagen fibrils will determine the orientation ofthe mineral crystals (Martin, R. B. et al. [1998] “Skeletal TissueMechanics”, Springer-Verlag Publishers, New York, N.Y.).

There are numerous biocompatible artificial bone substitutes currentlyon the market. Of these substitutes, none successfully mimics thecomposite or microstructure of bone. For example, man-made ceramiccomposites have some of the desired properties of natural bone (such asmatching of modulus), but are notoriously brittle and prone to cracking.By contrast, biological ceramics like bone and teeth resist cracking,with a high toughness and stiffness. It is the nanostructuredarchitecture that leads to mechanical properties that are unique tobone, which are not readily duplicated by polymers (which are not strongor stiff enough), or ceramics (which are brittle and lack toughness, andusually not bioresorbable). These mechanical properties are importantbecause of the body's natural repair processes, in which bone is aliving tissue and the cells respond according to the stresses they sensein their surrounding tissue (according to Wolf's Law). If an implantmaterial has too high of a modulus (stiffness), the cells tend to resorbthe surrounding bone due to the phenomenon of stress shielding (thestiffer material carries more of the load than the surrounding bone).

A logical choice of materials for a synthetic bone substitute would be acollagen-hydroxyapatite composite; indeed, many have attempted tomineralize collagen in vitro, but the preparation of such a compositehas been limited by the ability to achieve the high mineral loading thatis attained biologically by intrafibrillar mineralization. An associatedperiodic contrast pattern is commonly observed by transmission electronmicroscopy (TEM) of collagen fibers (Carter, J. G. [1990] SkeletalBiomineralization: Patterns, Processes and Evolutionary Trends, Volume1, Van Nostrand Reinhold Publishers, New York, N.Y.; Hodge, A. J. et al.[1963] “Recent studies with the electron microscope on orderedaggregates of the tropocollagen molecule”, in Aspects of ProteinStructure, Ramanchandran, G. N. (ed.), pp. 289-300, Academic Press,London, England; Katz, E. P. et al. [1989] Connect. Tissue Res.,21:49-159). From tomographic imaging of naturally mineralizing turkeytendon (which is considered a model of secondary bone formation), thereis evidence that the hydroxyapatite crystals first appear within thehole zones of collagen, and then spread throughout the fibrils, leadingto the array of iso-oriented nanocrystals of highly organizedhydroxyapatite [HAP] embedded within the organic matrix (Landis, W. J.et al. [1993] Structural Biology, 110:39-54; Landis, W. J. et al. [1991]Connect. Tissue Res., 25:181-196). Alternatively, there has beenevidence that the collagen fibers contain an amorphous substance duringthe early stages of bone formation, referred to by Bonnuci as an“inorganic substance in bands” (ISBs), which then crystallizes into themore commonly observed platy crystals (Bonnuci, E. Calcification inBiological Systems [1992] CRC Press Boca Raton, Fla.).

From a materials engineering perspective, the nanostructure of bone isintriguing and can be difficult to define. For example, it is not clearwhether bone is more accurately characterized as apolymer-fiber-reinforced ceramic-matrix composite or aceramic-nanoparticle-reinforced polymer-matrix composite. The two phasesare so intimately linked that the mechanical properties are distinctlydifferent than ceramics or polymers, and therefore are difficult toreproduce. To date, scientists do not have a complete understanding ofhow bone is formed, even at this most basic level of structure. However,it is likely that the nanostructured architecture plays a role in thetoughness of bone. Obviously, cellular control is important inbiomineralization, and in the case of bone, helps to build itshierarchical structure (i.e., lamellae and osteons), but even thephysicochemical mechanism for generating this nano-architecture has notbeen elucidated. Because intrafibrillar mineralization does not occursimply by attempting to crystallize collagen in vitro usingsupersaturated solutions of HAP (crystals only nucleate heterogeneouslyon the surface of the collagen fibers), it is generally assumed thatnucleating proteins must be present within the gaps of the collagenfibrils.

It is understood within the biomineralization community that acidicproteins can act as inhibitors to crystal nucleation or growth (Addadi,L. et al. [1987] Proc. Natl. Acad. Sci. USA, 84:2732-2736; Addadi, L. etal. [1992] Angew. Chem. Int. Ed. Engl. 31:153-169; Mann, S. et al.[1983] Structure and Bonding, 54:125-174; Mann, S. et al. [1989]“Crystallochemical Strategies in Biomineralization” inBiomineralization-Chemical and Biochemical Perspectives. Mann, S., Webb,J., and Williams, R. J. P. (eds.), 33-62 (VCH Publishers, N.Y., N.Y.)).In the case of crystal growth, it has been shown that selectiveinhibition of growth along stereospecific crystallographic planes canlead to a change in crystal morphology (Addadi, L. et al. Angew. Chem.Int. Ed. Engl., 24:466-485). In at least a few cases, acidic proteinshave been shown to promote crystal nucleation (Addadi, L. et al. [1987]Proc. Natl. Acad. Sci. USA, 84:2732-2736; Greenfield, E. M. et al.[1984] Amer. Zool., 24:925-932). It has also been shown that if theinhibitory action of a macromolecule is not complete, certain conditionslead to the induction (stabilization) of an amorphous liquid-phaseprecursor (Gower, L. B. et al. [2000] J. Crystal Growth,210(4):719-734), which can have a profound consequence on crystalmorphology since transformation of an amorphous precursor does notproceed via the same mechanism as traditional solution crystal growth(Mann, S. et al. [1989] “Crystallochemical Strategies inBiomineralization” in Biomineralization-Chemical and BiochemicalPerspectives. Mann, S., Webb, J., and Williams, R. J. P. (eds.), 33-62(VCH Publishers, N.Y., N.Y.)). Certain features of this polymer-inducedliquid-precursor (PILP) process suggest that this mechanism may occurduring morphogenesis of calcium carbonate biominerals in invertebrates(Gower, L. A. [1997] “The Influence of Polyaspartate Additive on theGrowth and Morphology of Calcium Carbonate Crystals,” Doctoral Thesis,Department of Polymer Science and Engineering, University ofMassachusetts at Amherst, 1-119).

It would be desirable to have the capability to synthetically prepare abone graft substitute that matches both the chemical and mechanicalproperties of bone. Such a material would be both load-bearing (with theappropriate modulus, strength, and toughness), yet bioresorbable toallow for the body's own tissue repair process to regenerate naturalbone.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns an organic/inorganic composite comprisingan inorganic mineral phase deposited onto and within an organic matrix,which is useful as a biomimetic substitute for bone and other tissues.The organic matrix is fluid-swellable, fibrous, and is penetrated by theinorganic mineral phase while the inorganic mineral phase is in the formof an amorphous polymer-induced liquid-precursor (PILP) phase.Optionally, while in the liquid-precursor phase, the inorganic mineralpenetrates and saturates the matrix, which can cause the matrix toswell. The fluid-swellable matrix can also include interstitial spacesand pores within the matrix structure, having the inorganic mineraldeposited therein.

Preferably, the fluid-swellable matrix of the composite is alongitudinally aligned fibrous material, with the inorganic mineraldeposited intrafibrillarly within the matrix, with the mineral phasealigned along the long axes of the fibers of the fluid-swellable matrix.More preferably, the organic substrate is collagen and the inorganicmineral is calcium phosphate, calcium carbonate, or a mixture thereof,wherein the inorganic mineral is deposited intrafibrillarly within thecollagen substrate. Examples of suitable calcium-containing inorganicminerals that can be used for the organic/inorganic composites of theinvention include, but are not limited to, calcium phosphate, calciumcarbonate, hydroxyapatite, strontium carbonate, calcium sulfate, calciumoxalate, calcium oxide, magnesium-bearing calcium carbonate orphosphate, calcium sulfate, calcium oxalate, and magnesium-bearingcalcium carbonate or phosphate, or any polymorphs of these minerals.

In another aspect, the subject invention concerns a process for makingthe composite described herein involving the inclusion of short-chainedacidic polymers to a supersaturated mineralizing solution, in order toinduce an amorphous liquid-phase precursor to the mineral, which is thenpulled by capillary action into interstices of the organic matrix (thusinfiltrating the organic substrate), and subsequently mineralizes viasolidification and crystallization of the precursor phase.

By using a PILP phase, the process of the present invention permitssuperior infiltration of an inorganic mineral phase (such as calciumphosphate or calcium carbonate) into an organic matrix (such ascollagen), closely mimicking the structure of natural bone, which is acomposite of collagen and calcium phosphate. In addition to having thecapability to be highly mineralized (thermogravimetric analysisindicates that the composites can contain 60% by weight mineral), theorganic/inorganic composite of the present invention is biocompatible,bioresorbable, and capable of load-bearing applications, such as use asa bone-graft substitute in critical-sized osseous defects, or jointreplacement (such as artificial hip replacement).

The organic substrate that is mineralized according to the process ofthe subject invention preferably comprises collagen fibrils. However,other fluid-swellable, fibrous materials, having organic molecules thatassemble into a secondary (e.g., supramolecular) fiber structure havinga high aspect ratio (length-to-diameter) sufficient to support alignmentof the mineral phase can be used.

The present invention further concerns a method of treating a patienthaving a bone defect by applying a composite described herein to thesite of the bone defect.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1B show scanning electron micrographs (SEMs) of “fibrous”biomineral morphologies that occur in the teeth of invertebrates andvertebrates. FIG. 1A shows that the rib region of a sea urchin tooth(Arbacia tribuloides) contains magnesium bearing calcium carbonate“rods”, 5-7 μm in diameter, embedded in an amorphous CaCO₃ matrix. FIG.1B shows that the ultrastructure of enamel from a rat incisor is morecomplex because it is “woven” into a cross-ply architecture by theameloblast cells. Of relevance is the non-equilibrium morphology of thecrystals, which in the vertebrates are polycrystalline bundles of HAP,rather than single-crystalline calcite rods, as in the urchin tooth.This further supports the assertion that the PILP process plays afundamental role in the morphogenesis of biominerals, both in thevertebrates and invertebrates.

FIGS. 2A-2C show SEM, transmission electron microscopy (TEM) and energydispersive spectroscopy (EDS) analyses of calcium phosphate PILP phaseprepared with CaCl₂, poly(vinyl phosphonic acid) (PVPA) andpolymer-assisted solution-phase (such as polyaspartic acid: PASP)additives. FIG. 2A shows a SEM micrograph of solidified precursordroplets. The droplets appear to have only partially coalesced and maynot have been as fluidic as is typical of the CaCO₃ PILP phase. FIG. 2Bshows a TEM micrograph of early stage solidified PILP droplets (averagesize≈200 nm). Light scattering studies have shown that the PILP droplets(for CaCO₃) start out approximately 100 nm in diameter and grow steadilyuntil they reach a size of a couple of microns (when they become visibleby optical microscopy). FIG. 2C shows an EDS of the sample shown in FIG.2A confirming that the PILP phase is composed of calcium and phosphate.The small C and O peaks suggest that carbonate may also be present,which is difficult to eliminate due to CO₂ in the atmosphere.

FIGS. 3A and 3B show SEMs of PILP-mineralized bovine collagen. Themineral precipitated predominantly as a coating on the fibers, but insome regions, as illustrated in FIGS. 3A and 3B, isolated platy tabletsdeposited along the fiber in a banded pattern. These bands of CaCO₃tablets (composition verified by EDS) are perpendicular to the long axisof the collagen fiber (indicated with an arrow), and appear to beassociated with the topography of the fiber (perhaps nucleated on thecrimps).

FIGS. 4A-4D show SEMs of mineralized CELLAGEN sponge. FIG. 4A shows thatthe sponge, as received (not mineralized), consists of an isotropic meshof type-I collagen fibers. FIG. 4B shows that the control reaction, inwhich collagen was mineralized with CaCO₃ without the addition ofpolymer, shows large rhombohedral crystals of calcite which nucleatedheterogeneously on the collagen. FIG. 4C shows that when the sponge wasmineralized using the PILP process, very different morphologies wereformed, in which patchy, thick calcitic films were deposited. FIG. 4Dshows that at higher magnification, it can be seen that the thick filmis actually composed of collagen fibers encased in mineral. Because theencasing mineral is solid, it does not dehydrate to as large an extentas the surrounding matrix of pure collagen upon vacuum treatment for SEManalysis; therefore, the film-like composite is much thicker than thesurrounding non-mineralized region, and appears as a step. It is alsoevident that the mineral provides some protection against beam damage,which was not the case for the un-mineralized region, in which a crackformed just below the mineralized patch. Note-the speckled appearance ofthe mineral is typical of PILP products, in which some precursordroplets do not fully coalesce to form smooth films (Gower, L. B. and D.J. Odom J. Crystal Growth 2000, 210(4):719-734; Gower, L. A. and D. A.Tirrell J. Crystal Growth 1998, 191(1-2):153-160).

FIGS. 5A and 5B show SEMs with banding patterns on mineralizedcollagens. FIG. 5A shows that this bovine collagen fiber was only partlymineralized, yet the fiber did not densify upon dehydration, suggestingthat some of the mineral had penetrated into the fiber, which holds theremaining non-mineralized sub-fibers in place (arrow). In both thebovine collagen and CELLAGEN sponge, banding patterns became apparentafter mineralization, which are not observed in the fibers as received(see FIG. 4A). In this case, however, the bands are not composed oftablets, but rather by a slight blebbing of the mineral coating (bottomright of fiber). The spacing of the bands is much smaller, at asub-micron size scale (unlike the dimensions of the collagen crimp shownfor the tablets in FIG. 3). FIG. 5B shows that blebbing of the smallcollagen sub-fibers in the CELLAGEN sponge is very pronounced in thisparticular region, to the point of having a disc-like appearance, inwhich the discs are oriented perpendicular to the long axis of thefibers. The banding pattern here is robust enough to measure; the bandsoccur at approximately every 380 nm, which is approximately six timesthe 64 nm spacing of type I collagen.

FIGS. 6A-6D show SEM micrographs of DAVOL Ultrafoam sponges underdifferent mineralization conditions. FIG. 6A shows DAVOL Ultrafoam, notreatment, as received showing pore size on the order of 2-100 μm indiameter. FIG. 6B shows DAVOL Ultrafoam mineralized in the absence ofpolymeric additives, in which 20 μm diameter calcite rhombohedralsnucleated on the surface of the sponge. FIG. 6C shows a partiallymineralized section of DAVOL Ultrafoam mineralized in the presence ofPAA (5100 MW, ALDRICH). FIG. 6D shows a fully mineralized section ofDAVOL Ultrafoam mineralized in the presence of PAA (5100 MW, ALDRICH).

FIG. 7 shows X-ray diffraction (XRD) analysis, with Line A showing thediffraction pattern of dehydrated collagen sponge held in distilledwater for three days, Line B showing the diffraction pattern of collagensponge mineralized without polymer for three days in a supersaturatedCaCO₃ solution, and Line C showing the diffraction pattern of collagensponge mineralized for three days in a supersaturated solution of CaCO₃containing acidic polymers. The characteristic calcitic peaks on Line Band Line C are in the same position. The peaks in Line C are muchbroader than those in Line B, suggesting that either there is strain inthe crystals, or the crystals are of much smaller size as compared tothose in Line B (Cullity, B. D., “Elements of X-ray Diffraction” [2001]Addison-Wesly Longman, N.Y.)

FIGS. 8A-8C show calcium phosphate mineralized CELLAGEN sponge etchedwith 0.1 N HCl for 15 minutes to remove surface encapsulating mineral.Results are similar to CELLAGEN sponge mineralized with CaCO₃.

FIGS. 9A-9D show TEM, Bright Field (BF) and electron diffractionanalyses of mineralized CELLAGEN experiments. FIG. 9A shows a BF imageof a CELLAGEN sponge stained with 1% phosphotungstic acid in 0.1M PBS. A67-70 nm characteristic collagen banding pattern is observed. FIG. 9Bshows a diffraction pattern of stained collagen. Observed pattern issimilar to transmitted beam pattern, as there are no diffraction spots.FIG. 9C shows a BF image of CELLAGEN sample mineralized with CaP mineralthrough the PILP mechanism. Notice the 50-70 nm round particles aroundand in the collagen fibril (arrows), which are believed to be PILPdroplets adsorbed to the collagen. Bar=100 nm. FIG. 9D shows a typicaldiffraction pattern of mineralized collagen fibril. Notice strongdiffraction peaks in the direction of the c-axis of the collagen fibril.

FIGS. 10A and 10B show SEM micrographs of type-I collagen spongemineralized with hydroxyapatite (HA) mineral via the PILP process. InFIG. 10A, each individual collagen fibril is encased in mineral. Bar=10μm. FIG. 10B shows high magnification of mineralized fibril bundlesobserved using FEG-SEM. The platy/needle-like crystals appear to beoriented along the long axis of the fibril bundle (arrow). Bar=100 nm.

FIGS. 11A-11C show TEM micrographs of a single collagen fibrilmineralized via a HA PILP phase. FIG. 11A shows BF image of mineralizedfiber. Bar=200 nm. FIG. 11B shows a selected area diffraction (SAD)pattern of the middle of the fiber in FIG. 11A, indicating that themineral is hydroxyapatite. Note that arcs for the (002) and (004) planesare oriented perpendicular to the long axis of the collagen fibril(marked with long arrow), indicating that the HA crystals are alignedwith their c-axes parallel to the fiber (in the [001] direction). FIG.11C shows a Weak-Beam Dark-Field TEM micrograph of the fiber in A,illuminating the crystals that generated diffraction from the (002)plane. Note the illuminated crystals are long and needle-like (or thinplates edge on) and oriented along the long axis of the collagen fibril.Bar=200 nm.

FIG. 12 shows SAD patterns derived from mineralized collagen samples.The left panel of FIG. 12 is the diffraction pattern from HA PILPmineralized collagen of the present invention. The right panel of FIG.12 is a SAD diffraction pattern from a 50 year old male femur (afterZiv, V., et al., Microscopy Research and Technique, 1996,33(2):203-213). Note how the patterns are identical, from the arcing ofthe (002) and (004) planes, which are aligned along the long axis of thecollagen fibril, to the (112) diffraction ring just past the (002)diffraction spot. Note-a diffraction pattern containing arcs indicatesslight mis-alignment of crystal planes, as compared to a spot patternthat is seen for single-crystalline materials.

FIGS. 13A-13C show electron micrographs of mineralized collagen fibrils.FIG. 13A shows an SEM of the mineralized collagen fibril imaged in theleft panel of FIG. 12. Note the non-descript surface features of thecollagen fibril. There appear to be no platy crystals on the surface,suggesting that the crystals illuminated by the PMDF TEM image in FIG.11C are within the fiber (i.e. the collagen fibril is intrafibrillarlymineralized). Bar=100 nm. FIG. 13B shows Energy Dispersive Spectroscopicanalysis of a point in the middle of the fibril in A. Note that thespectrum has been increased to highlight the Ca, P and O peaks whichwere drowned out by the large peaks from the Al SEM stub and the Cu TEMgrid. The large C peak is an artifact of the amorphous carbon coatingprocess used to prevent charging in SEM. FIG. 13C shows a Bright FieldTEM image of a mineralized collagen fibril. Platy crystals can be seenalong the long axis of the collagen fibril. Note the 64 nm bandingpattern of type-I collagen fibril that appears perpendicular to the longaxis of the collagen fibril (highlighted by the arrow). Bar=100 nm.

FIG. 14 shows an SEM of a SCOTCH BRITE sponge that has not beenintroduced to mineralizing solutions. This household sponge hasmicropores on the order of 0.75 microns-2.0 microns in diameter.

FIG. 15 shows an SEM of a CELLAGEN sponge (reconstituted bovine Type-Icollagen) that has been mineralized using the process of the subjectinvention. The fibers are collagen and the mounds are collagen fiberscoated in calcium carbonate.

FIG. 16 shows an SEM of a SCOTCH BRITE sponge that has been mineralizedusing the process of the subject invention. The sponge has been coatedwith non-equilibrium morphology calcium carbonate, thus clogging some ofthe pores.

FIG. 17 is an SEM of a CELLAGEN sponge that has been mineralized usingthe process of the subject invention. The sample was mineralized fourtimes. Specifically, the sample was run for three days and themineralizing solution was subsequently refreshed (the equivalent of onemineralization). The sample was then placed in a 0.1N HCl solution toetch away the surface coating of calcium carbonate. The large bundle offibers in the middle of the picture shows how the whole bundle may havebeen coated (hence, the end which still seems to be coated) and the acidetch preferentially left behind bands of calcium carbonate.

FIG. 18 shows an SEM of a CELLAGEN sponge that has been mineralizedusing the process of the subject invention. The sample was mineralizedfive times. Specifically, the sample was run for three days and themineralization solution was subsequently refreshed (the equivalent ofone mineralization). The sample was then placed in a 0.1 N HCl solutionto etch away the surface coating of calcium carbonate. Again, all of thefibers appear to have been coated and the acid etch preferentiallyetched away the excess calcium carbonate, leaving behind banded calciumcarbonate that was protected from the etchant by the surroundingcollagen fiber.

FIG. 19 shows an SEM of a CELLAGEN sponge that has been mineralizedusing the process of the subject invention. The sample was mineralizedfive times. Specifically, the sample was run for three days and themineralization solution was subsequently refreshed (the equivalent ofone mineralization). The sample was then placed in a 10% bleach solutionto etch away the organic material, specifically collagen. With thecollagen removed, it can be seen that the calcium carbonate that remainshad totally infiltrated the collagen (the perpendicular bands of mineralspan completely across the pre-existing fibers).

FIGS. 20A and 20B shows SEM images of mineralized CELLAGEN scaffolds(with polymer) after exposure to mineralized solution. FIG. 20A showsosteoblasts adhered to mineralized scaffold at a magnification of 150×.FIG. 20B shows an adhered osteoblast producing mineralization nodules ata magnification of 1000×.

FIG. 21 shows the number of cells adhering to the three differentscaffold types after a three and a half hour exposure. The three typesof scaffolds include A) unmineralized with 9,658 cells; B) mineralizedwithout polymer with 18,223 cells; and C) mineralized with polymer with20,296 cells.

FIGS. 22A-22E show materials and methods for producing individuallymineralized films of the present invention.

FIGS. 23A-23C show mineralized films of the present invention, which canbe adhered to one another in an alternating orientation, to form across-ply architecture.

DETAILED DISCLOSURE OF THE INVENTION

The composite of the present invention comprises an organic substrateand an inorganic mineral phase, useful as a biomimetic material of boneor other hard tissue. Thus, the composite of the subject invention is anorganic/inorganic composite that is highly mineralized, with its organicand inorganic components intimately associated in a manner mimicking thestructure of natural bone. The organic substrate is composed of afluid-swellable, fibrous matrix, which is penetrated by the inorganicmineral phase while the inorganic mineral is in the form of a transientpolymer-induced liquid-precursor phase (PILP) that subsequentlysolidifies. Advantageously, once solidified, the inorganic mineral isoriented such that it is aligned along (e.g., running substantiallyparallel with) the long axes of the fibers of the organicfluid-swellable, fibrous matrix, thereby closely mimicking the structureof natural bone.

Optionally, while in the liquid precursor phase, the inorganic mineralpenetrates and saturates the matrix, which can cause the matrix toswell. The fluid-swellable, fibrous matrix can also have interstitialspaces and pores, having the inorganic mineral deposited therein.

The organic substrate that is mineralized with an inorganic phaseaccording to the process of the subject invention is fibrous. In oneembodiment, the fibrous organic substrate comprises collagen fibrils asa substantially parallel-fibered matrix. In one embodiment, the fibrousorganic substrate is collagen. However, other fluid-swellable, fibrousmaterials, having organic molecules that assemble into a secondary(e.g., supramolecular) structure having a high aspect ratio (i.e., afiber) sufficient to support alignment of the mineral phase can be used.For example, elastin, cellulose, chitin, chitosan, and/or thepeptide-amphiphile nanofibers described by Hartgerink et al.(Hartgerink, J. D. et al., Science, 2001, 294:1684-1688; Hartgerink, J.D. et al., PNAS, 2002, 99(8):5133-5138) can be mineralized using themethods of the present invention. The inorganic phase is preferablycalcium phosphate, calcium carbonate, or a mixture thereof. Examples ofsuitable calcium-containing inorganic minerals that can be used for theorganic/inorganic composites of the invention include, but are notlimited to, calcium phosphate, calcium carbonate, hydroxyapatite,strontium carbonate, and calcium sulfate, calcium oxalate, calciumoxide, magnesium-bearing calcium carbonate or phosphate, or anypolymorphs of these minerals.

As used herein, the term “fibrous” is intended to refer to organicfluid-swellable matrices having at least some fiber (high aspect ratio)component. The fibers can be of various lengths, diameters, andorientations (e.g., parallel, mesh, random). The terms “fibers” and“fibrils” are used interchangeably herein to refer to filamentous orthread-like matter and any sub-fibers that contribute to a larger fiber.The fibers can be produced by self-assembly or directed assembly ofmolecules through synthesis procedures. Other fibrous matrices can befound, for example, in Vincent, J. (Structural Biomaterials, PrincetonUniversity Press, Princeton, N.J., 1990, Chapter 3, pp. 73-91).

The composite of the subject invention has similar mechanical, chemical,and biological properties to normal human bone tissue; thus,representing a biomimetic of bone (emulating the structure andmechanisms of the biological tissue), without the shortcomings ofcurrent artificial bone-replacement materials. For example, oncesolidified and crystallized, the inorganic mineral phase of thecomposite of the present invention lacks the facets typically present inother artificially mineralized collagen composites. Therefore, thecomposite of the present invention more closely resembles the patternsof mineral deposition found in nature, such as in human bone. Inaddition, the composite of the subject invention does not have thedisadvantages associated with using donor tissue, such as diseasetransfer, rejection, unknown resorption rate, and scarcity.

In another aspect, the subject invention concerns a process for makingthe composite described herein involving the inclusion of one or moreacidic polymers to a supersaturated mineralizing solution, in order toinduce an amorphous liquid-phase precursor to the mineral, which is thenabsorbed by capillary action into the fluid-swellable, fibrous matrix ofthe organic substrate, and subsequently solidifies and crystallizes intothe mineral phase. Preferably, the acidic polymer is a short-chainedacidic polymer. The process of the subject invention, which uses apolymer-induced liquid precursor (PILP), permits superior infiltrationof the mineral phase into the organic phase, such that the mineral phaseis intimately associated with the organic matrix. In composites havingan organic matrix comprising fibrils, the mineral phase can beassociated with the organic matrix to the extent that nanocrystals areembedded within the interstices of the fibrils. Thus, as used herein,the term “liquid-precursor” or “liquid-phase mineral precursor” refersto an inorganic mineral phase that is sufficiently fluid to infiltrate,or be absorbed by, the fluid-swellable matrix.

The process of the subject invention comprises contacting an acidicpolymer, such as poly-L-aspartic acid and/or polyacrylic acid, with amineralizing solution of a calcium-containing mineral, thereby forming aliquid-phase mineral precursor; and contacting an organic substrate withthe liquid-phase mineral precursor, wherein the organic substrate iscollagen or another fluid-swellable, fibrous material (such aspeptide-amphiphile nanofibers). The calcium-containing mineralizingsolution can contain, for example, calcium salt, calcium chloride,and/or calcium phosphate, or other calcium-containing minerals. Theliquid-phase mineral precursor is fluidic and amorphous such thatdroplets of the precursor are drawn into the organic substrate bycapillary action. The droplets coalesce into a continuous coating andare preferentially absorbed into any interstices present within theorganic substrate. Advantageously, where the organic substrate iscollagen, the liquid-phase mineral precursor is drawn into the gaps andgrooves of the collagen fibrils. As used herein, the term “gap” refersto the interstitial space between abutting ends of fibrils. Contiguousgaps among adjacent fibrils are referred to herein as “grooves”.

The polymeric additive stabilizes the amorphous precursor to the mineralthat is in the form of a liquid or liquid-like viscosity. Uponsolidification of the precursor, non-equilibrium (non-faceted) crystalmorphologies are generated that are distinct from the crystals producedby other solution crystallization processes carried out under similarconditions without the polymeric additive. In particular, because of thefluidic nature of the precursor, many of the highly unusual morphologiesassociated with biominerals (such as biominerals “molded” withinvesicular compartments or “extruded” into fibers), can readily beaccomplished using the process of the present invention.

After having coated and infiltrated the organic substrate, the amorphousmineral precursor solidifies and crystallizes, and the overall compositedensifies. In the case of collagen, the present inventors have observedthat disk-shaped crystals can be left periodically spaced within thefibers at approximately 380 nm intervals, which correlates toapproximately six times the natural gap spacing of collagen. Presumably,the gaps have aligned across the fiber to form grooves capable ofmineralizing, or the mineral diffuses throughout the interstitial spaceof the matrix, and is not restricted to just the hole zones.Interstitial space refers to the space between macromolecular chains, orin the case of collagen, the space between the staggered array oftropocollagen rods. Therefore, in the case where calcium carbonate isthe inorganic mineral, the result is a nanostructured composite withcalcitic nanocrystals embedded within the collagen fibrils, in a similarfashion to natural bone.

One or more steps of the process of the subject invention can berepeated. Hence, sequential loading of the organic substrate with theinorganic mineral can be carried out, resulting in various degrees ofmineralization of the organic substrate. The extent of mineralizationcan be determined using methods of polymer matrix analysis known tothose of ordinary skill in the art, such as thermogravimetric analysis(TGA) and ash fractionation of the inorganic and organic phases.Preferably, the composite of the subject invention comprises about 25%to about 40% organic matrix (such as collagen or other fluid-swellable,fibrous material) and about 35% to about 50% mineral phase (such ascalcium phosphate, calcium carbonate, or a mixture thereof), by volume.The remaining component (about 10% to about 40%) can be water andnon-collagenous (glyco)proteins, for example. More preferably, thecomposite of the subject invention comprises about 30% to about 34%organic substrate, and about 40% to about 45% mineral phase, by volume.Most preferably, the composite of the subject invention comprises about32% organic substrate, about 43% mineral phase, and about 25% water, byvolume, similar to the composite of natural bone.

Using a polymer-induced liquid phase precursor, the process andcomposites of the present invention are distinguishable from knownmethods for mineralization of reconstituted collagen that yield onlyequilibrium calcite rhombohedral crystals that nucleate heterogeneouslyand grow only on the exterior surface of the fibers. Mineralizationusing the process of the subject invention permits infiltration of themineral into the matrix structure of the organic substrate, as evidencedby the banding of the collagen fibers, which consists of calcitic diskslying perpendicular to the long axis of the collagen fibers. Thisbanding pattern is somewhat periodic and occurs with a spacing ofapproximately 250 nm to 300 nm, which roughly corresponds to six timesthe natural 64 nm spacing of hole zones within biogenic tropocollagen(the spacing is likely disturbed due to reconstitution of the commercialType-1 collagen; alternatively, the spacing may arise fromdiffusion-limited exclusion of impurities (e.g., collagen and acidicpolymer) during the crystallization process). Without being bound bytheory, it is presumed that the PILP mineralization process results fromthe ion-binding affinities of the short-chained acid polymer inconjunction with water retention and nucleation inhibition, such that ametastable ionic liquid-phase precursor is generated.

The liquid-phase precursor of the inorganic mineral can be producedusing a variety of methods. For example, synthesis of a liquid-phaseprecursor of calcium carbonate has been described previously (Gower, L.B. and D. J. Odom J. Crystal Growth 2000, 210(4):719-734; Gower, L. A.[1997] “The Influence of Polyaspartate Additive on the Growth andMorphology of Calcium Carbonate Crystals,” Doctoral Thesis, Departmentof Polymer Science and Engineering, University of Massachusetts atAmherst, 1-119; Gower, L. A. and D. A. Tirrell J. Crystal Growth 1998,191(1-2):153-160), where the vapor diffusion of the decompositionproducts of crushed ammonium carbonate (NH₄)₂CO₃ into a solutioncontaining calcium chloride (CaCl₂) and one or more short-chain acidicpolymer additives. Alternative methods can be used to gradually raisethe supersaturation of the crystallizing solution, including directaddition of a carbonate containing solution to the calcium containingsolution; or the escape of carbon dioxide gas from a saturated calciumbicarbonate solution (which produced by bubbling carbon dioxide into anaqueous solution containing the calcium carbonate salt). Supersaturationcan also be raised using temperature, pH, or removal of inhibitoryspecies. Similar methods can be used for the calcium phosphate system,although higher temperature (37° C.) is more favorable for decompositionof ammonium phosphate (or any derivations of ammonium phosphate, such asammonium phosphate dibasic). In addition, the phosphate counterion canbe produced using enzymes that cleave phosphate-containing moieties.According to the mineralization process of the subject invention, theorganic substrate can then be contacted with the liquid-phase precursorof the inorganic mineral, such as calcium phosphate, calcium carbonate,hydroxyapatite, strontium carbonate, calcium sulfate, calcium oxalate,calcium oxide, magnesium-bearing calcium carbonate or phosphate, or anypolymorphs of these minerals.

The process of the subject invention can be carried out under a varietyof conditions. For example, in the case of an aqueous system, theprocess can be carried out at a temperature of about 0° C. to about 100°C. Preferably, the process is carried out at about 37° C., to matchphysiological conditions. The process can be carried out at a pH in therange of about 5 to about 10. Preferably, the process is carried out ata pH of about 7.0 to about 7.8 and 1 atm. More preferably, the processis carried out at a pH of about 7.4.

The type of reaction vessel or vessels utilized for preparing thecomposite of the subject invention, or their sizes, are not critical.Any vessel or substrate capable of holding or supporting the organic andinorganic phases so as to allow the reaction to take place can be used.Preferably, the supersaturation is gradually increased, allowing timefor the acidic polymer to induce and stabilize the liquid-phase mineralprecursor. It should be understood that, unless expressly indicated tothe contrary, the terms “adding”, “contacting”, “mixing”, “reacting”,and grammatical variations thereof, are used interchangeable to refer tothe mixture of reactants of the process of the present invention (e.g.,acidic polymer additives, calcium-containing solution, and so forth),and the reciprocal mixture of those reactants, one with the other (i.e.,vice-versa).

Where collagen is used as the organic phase of the composite of thesubject invention, the collagen can be obtained from mineralized orunmineralized sources. Preferably, the collagen is obtained from anunmineralized collagen source. Therefore, the collagen may come frombiological sources such as bone, tendons, skin, or the like. Preferably,the collagen involves a combination of three strands of α2 collagenchains. The collagen can be obtained from a young, intermediate, ormature animal, such as mammals or avians. Examples of convenient animalsources of collagen include chicken, turkey, bovine, porcine, or equinesources. The collagen can also be recombinant collagen, or artificiallysynthesized. Collagen of various types can be used as any of collagentypes I-XX, or combinations thereof.

Fluid-swellable (and particularly water-swellable), fibrous materialsother than collagen can also be utilized as organic substrates withinthe composites of the subject invention. For example, fibrouspolysaccharides (such as chitin and cellulose) can be used. Otherexamples of fluid-swellable materials appropriate as organic substratesinclude elastin, chitosan, and peptide nano-fibers. Any fluid-swellable,fibrous material can be utilized as the organic substrate, provided thatthe material can swell sufficiently to absorb the liquid-phase mineralprecursor and support alignment of the mineral phase along the long axesof the fibers. Preferably, the fluid-swellable, fibrous material isbioresorbable and/or biocompatible so that cells can infiltrate and growwithin the composite and eventually remodel it into natural bone tissue.However, bioresorbability may not be necessary if the composite materialis biocompatible and has sufficient mechanical properties for long-termor permanent application.

The fluid-swellable, fibrous matrix can be surface modified before,during, or after mineralization using any of a variety of means known inthe art, such as plasma treatment, etching, ion implantation, radiation,electron beam, chemical functionalization, grafting,photopolymerization, adsorption, or combinations thereof.

One or more of a variety of acidic polymers, such as acidicshort-chained polymers, can be utilized to initiate the amorphousliquid-phase mineral precursor, including different polymers andbiological materials. Polyacrylic acid (PAA), polymethacrylates (PMA),sulfonated polymers, phosphorylated proteins, peptides and polymers,sulfated glycoproteins, polyaspartic acid, polyglutamic acid,polyaspartate, polyvinyl phosphate, and blends or copolymers of thesematerials, individually and in mixtures, can be utilized to induce theliquid-phase separation, for example. A range of polymer molecularweights can be suitable if the other variables of the crystallizingconditions are appropriately modified to generate the PILP phase.Preferably, molecular weights in the range of 2,000 to 15,000 g/molenhance the ability to induce formation of the precursor.

The desired hardness of the composite of the subject invention can beachieved by varying the weight ratio of the organic phase (such ascollagen) and the inorganic phase (such as calcium phosphate and/orcalcium carbonate), and the physical nature of the phases (such asdegree of crystallinity, density, fibrillar structure of matrix, etc.).

The composite of the subject invention can be applied as a film orcoating on a substrate. The substrate can be composed of any material,such as metal, polymer, and/or ceramic materials (such as hip joints,knee joints, dental implants, spinal fusion cages, and bone fillers).The composite can also be formed by sequential formation of layers, inwhich the orientation of the polymer in adjacent layers may or may notbe controlled, similar to a laminated composite.

The present invention further concerns a method of treating a patienthaving a bone defect by applying a composite described herein to thesite of the bone defect. As used herein, the term “patient” refers toany human or non-human animal suffering from a bone defect. According tothe method of the subject invention, a therapeutically effective amountof the composite can be applied at the site of a bone defect topartially or fully restore structural integrity to the bone. Onceapplied, the composite of the subject invention can function as a filler(or partial filler) or plug, to mend the bone defect. The amount to beapplied will depend upon the size and nature of the bone defect, and theclinical outcome that is sought. The composite can be applied in amalleable form, for example, as a paste or putty, such that theadministered composite takes the shape of the bone defect.Alternatively, the composite can be molded pre-cast into a desired shape(such as the shape of the defect) using polymer composite moldingmethods known to those of ordinary skill in the art, and the moldedcomposite can be administered as a solid or semi-solid article. Thus,the size, volume, thickness, and shape of the molded article can becontrolled, as desired. The composite can be applied in particulateform. According to the method of the subject invention, the compositecan be applied so that it directly contacts existing bone adjacent to,or defining, the bone defect site, or the composite can be contactinganother implant, or both.

The composite of the subject invention can be applied to the bone defectsite as a liquid. Once applied, with a syringe for example, the liquidcomposite can coagulate or cure (“set”) shortly after application toform a solid.

The composite of the subject invention can be used as a vehicle for thein situ delivery of biologically active agents. The biologically activeagents incorporated into, or included as an additive within, thecomposite of the subject invention can include, without limitation,medicaments, growth factors, vitamins, mineral supplements, substancesused for the treatment, prevention, diagnosis, cure or mitigation ofdisease or illness, substances which affect the structure or function ofthe body, or drugs. The biologically active agents can be used, forexample, to facilitate implantation of the composite into a patient andto promote subsequent integration and healing processes. The activeagents include, but are not limited to, antifungal agents, antibacterialagents, anti-viral agents, anti-parasitic agents, growth factors,angiogenic factors, anaesthetics, mucopolysaccharides, metals, cells,and other wound healing agents. Because the processing conditions can berelatively benign (physiological temperature and pH), live cells can beincorporated into the composite during its formation, or subsequentlyallowed to infiltrate the composite through tissue engineeringtechniques.

As indicated above, cells can be seeded onto and/or within theorganic/inorganic composites of the present invention. Likewise, tissuessuch as cartilage can be associated with the composites prior toimplantation within a patient. Examples of such cells include, but arenot limited to, bone cells (such as osteoclasts, osteoblasts, andosteocytes), blood cells, epithelial cells, neural cells (e.g., neurons,astrocytes, and oligodendrocytes), and dental cells (odontoblasts andameloblasts). Seeded cells can be autogenic, allogenic, or xenogenic.Seeded cells can be encapsulated or non-encapsulated.

Examples of antimicrobial agents that can be used in the presentinvention include, but are not limited to, isoniazid, ethambutol,pyrazinamide, streptomycin, clofazimine, rifabutin, fluoroquinolones,ofloxacin, sparfloxacin, rifampin, azithromycin, clarithromycin,dapsone, tetracycline, erythromycin, cikprofloxacin, doxycycline,ampicillin, amphotericine B, ketoconazole, fluconazole, pyrimethamine,sulfadiazine, clindamycin, lincomycin, pentamidine, atovaquone,paromomycin, diclarazaril, acyclovir, trifluorouridine, foscarnet,penicillin, gentamicin, ganciclovir, iatroconazole, miconazole,Zn-pyrithione, and silver salts, such as chloride, bromide, iodide, andperiodate.

Growth factors that can be incorporated into the composite of thepresent invention include, but are not limited to, bone growth factors(e.g., BMP, OP-1) basic fibroblast growth factor (bFGF), acidicfibroblast growth factor (aFGF), nerve growth factor (NGF), epidermalgrowth factor (EGF), insulin-like growth factors 1 and 2 (IGF-1 andIGF-2), platelet-derived growth factor (PDGF), tumor angiogenesis factor(TAF), vascular endothelial growth factor (VEGF), corticotropinreleasing factor (CRF), transforming growth factors alpha and beta(TGF-α and TGF-β), interleukin-8 (IL-8), granulocyte-macrophage colonystimulating factor (GM-CSF), the interleukins, and the interferons.

Other agents that can be incorporated into the composite of the subjectinvention include acid mucopolysaccharides including, but not limitedto, heparin, heparin sulfate, heparinoids, dermatan sulfate, pentosanpolysulfate, chondroitin sulfate, hyaluronic acid, cellulose, agarose,chitin, dextran, carrageenin, linoleic acid, and allantoin.

Proteins that can be incorporated into, or included as an additivewithin, the composite of the subject invention include, but are notlimited to, collagen (including cross-linked collagen), fibronectin,laminin, elastin (including cross-linked elastin), osteopontin,osteonectin, bone sialoproteins (Bsp), alpha-2HS-glycoproteins, boneGla-protein (Bgp), matrix Gla-protein, bone phosphoglycoprotein, bonephosphoprotein, bone proteoglycan, protolipids, bone morphogeneticprotein, cartilage induction factor, platelet derived growth factor andskeletal growth factor, enzymes, or combinations and biologically activefragments thereof. Other proteins associated with other parts of humanor other mammalian anatomy can be incorporated or included as anadditive, include proteins associated with cartilage, such aschondrocalcining protein, proteins associated with dentin, such asphosphoryin, glycoproteins and other Gla proteins, or proteinsassociated with enamel, such as amelogenin and enamelin. Agentsincorporated into the composite of the subject invention may or may notfacilitate or enhance osteoinduction. Adjuvants that diminish an immuneresponse can also be used in conjunction with the composite of thesubject invention.

The biologically active agents can first be encapsulated intomicrocapsules, microspheres, microparticles, microfibers, reinforcingfibers and the like to facilitate mixing and achieving controlled,extended, delayed and/or sustained release. Encapsulating thebiologically active agent can also protect the agent against degradationduring formation of the composite of the invention.

Additionally, the biologically active agents can be pendantly attachedto the organic or inorganic phase. The attachment can be facilitatedthrough covalently linking the agent to the organic or inorganic phase,or through the use of hydrogen bonding.

In a preferred embodiment of the invention, the biologically activeagent is controllably released into a mammal when the composite of theinvention is implanted into a mammal, due to bioresorption relying onthe time scale resulting from cellular remodeling. Preferably, thecomposite of the subject invention is used to replace an area ofdiscontinuity in the bone tissue in the mammalian body. The area ofdiscontinuity in the bone can be as a result of trauma, disease, geneticdefect, tumor, or surgery, for example.

The composite of the subject invention can be formulated into a varietyof shapes suitable for its function as a bone graft substitute, such asa plate, pin, rod, screw, anchor, tack, arrow, staple, button, or otherregular or irregular shape. The composite of the present invention canbe formulated as a three-dimensional scaffold and, optionally, seededwith one or more cell types for implantation within a patient. Forexample, a highly mineralized, collagen/hydroxyapatite composite with ananostructured architecture similar to parallel-fibered compact bone canbe fabricated using the PILP process of the present invention. Using themethod of the present invention, mineralized scaffolds with thecomposition and mechanical properties of bone can be produced. Thesescaffolds can exhibit osteoconductive properties that promote rapidinfiltration of cells into the implant material for neovascularizationand osseous ingrowth. Hence, the organic/inorganic composites producedby the methods of the present invention can be sufficientlyosteoconductive, load-bearing, and bioresorbable so as to replace thecurrent “gold standard”, which requires harvesting of autogenous bonetissue.

The term “intrafibrillar”, as used herein, is used to specify thelocation of the crystallites, which has been described as a“deck-of-cards” arrangement of iso-oriented nanocrystals that areembedded within any interstitial spaces within the organic matrix. Forexample, in the case of HA, the platy/needle-like crystals are orientedwith the [001] direction of the crystal aligned parallel to the longaxis of the collagen fibril. In the case of collagen, these interstitialspaces include the gaps and grooves of the assembled collagen fibrils(Traub, W. et al. [1992] Matrix, 12:251-255; Weiner, S. et al. [1991]“Organization of Crystals in Bone”, in Mechanisms and Phylogeny ofMineralization in Biological Systems, Suga, S. and Nakahara, H. (eds.),pp. 247-253; Traub, W. et al., [1989] Proc. Natl. Acad. Sci.,86:9822-9826, Springer-Verlag Publishers, New York, N.Y.). Thus, as usedherein, intrafibrillar means that the amorphous precursor penetrates thefluid-swellable, fibrous matrix of the organic substrate, and mineralcrystals grow on and within the matrix structure, and on and within anyunderlying substructure, such as fibers. The amorphous mineral phasepenetrates the fibers (and fibrils, if present) of the fibroussubstrate, and mineral crystals grow on and within the fibers (andfibrils, if present) of the organic substrate. As used herein, the term“interfibrillar” means that the mineral crystals grow within thefluid-swellable matrix, but do not necessarily penetrate the interior ofthe fibrillar substructure generated from the self-assembly of theorganic molecules (e.g., tropocollagen molecules).

The term “bone defect”, as used herein refers to any bone deficientregion, such as a void, gap, recess, or other discontinuity in the bone.The bone defect can be artificially or naturally established, and canoccur due to disease or trauma, for example. Thus, the bone defect canoccur as a consequence of pathologic, inflammatory, or tumor diseases,surgical interventions, congenital defects, or bone fractures, and thelike. For example, in the case of certain diseases, such as bone tumors,the bone defect is artificially established by removing the tumortissue. Thus, according to the method of the subject invention, thecomposite can be applied, for example, to repair periodontal defects,for craniofacial reconstruction, joint reconstruction, fracture repair,to conduct orthopedic surgical procedures, and spinal fusion, forexample. The term “bony defect” is also intended to include anatomicalsites where augmentation to a bony feature is desired by the patient inthe absence of disease or trauma, such as in elective cosmetic surgery.Thus, the “defect” can be one that is subjectively perceived by thepatient, and where augmentation of the bone deficient region is desired.

The term “biocompatible”, as used herein, means that the material doesnot elicit a substantial detrimental response in the host. It should beappreciated that when a foreign object is introduced into a living body,that the object may induce an immune reaction, such as an inflammatoryresponse that will have negative effects on the host. As used herein,the term “biocompatible” is intended to include those materials thatcause some inflammation and/or tissue necrosis, provided that theseeffects do not rise to the level of pathogenesis. Various assays knownin the art can be utilized to assess biocompatibility. For example, thefollowing assays can be conducted to determine biocompatibility of cellsto the composites: alkaline phosphatase activity (e.g., using Diagnostickit 245, SIGMA) for a particular cell phenotype (e.g., osteoblast),methylthiazol tetrazolium (MTT) proliferation assay (Zund G. et al.[1999] European Journal Cardiothoracic Surgery 15:519-524; Deng Y. etal. [2002] Biomaterials 23:4049-4056), histology/histomorphometry, andSEM analysis. Response variables for subsequent statistical analysiswould include, for example, cell count, alkaline phosphatase expression,cell distribution, and cell/tissue penetration.

The term “biodegradable”, as used herein, means capable of beingbiologically decomposed. A biodegradable material differs from anon-biodegradable material in that a biodegradable material can bebiologically decomposed into units which may be either removed from thebiological system and/or chemically incorporated into the biologicalsystem.

The term “bioresorbable”, as used herein, refers to the ability of amaterial to be resorbed in vivo. “Full” resorption means that nosignificant extracellular fragments remain. The resorption processinvolves elimination of the original implant materials through theaction of body fluids, enzymes, or cells. Resorbed calcium carbonatemay, for example, be redeposited as bone mineral, or by being otherwisere-utilized within the body, or excreted. “Strongly bioresorbable”, asthe term is used herein, means that at least 80% of the total mass ofmaterial implanted is resorbed within one year.

The term “polymorph”, as used herein, refers to inorganic minerals thatare identical chemical compositions but different crystal structures.

The terms “comprising”, “consisting of”, and “consisting essentially of”are defined according to their standard meaning and may be substitutedfor one another throughout the instant application in order to attachthe specific meaning associated with each term.

Materials and Methods

Polymer-Induced Liquid-Precursor (PILP) Process. The preparation of aPILP phase has been described previously (Gower, L. B. and D. J. Odom J.Crystal Growth 2000, 210(4):719-734; Gower, L. A. [1997] “The Influenceof Polyaspartate Additive on the Growth and Morphology of CalciumCarbonate Crystals,” Doctoral Thesis, Department of Polymer Science andEngineering, University of Massachusetts at Amherst, 1-119; Gower, L. A.and D. A. Tirrell J. Crystal Growth 1998, 191(1-2):153-160). The generalprocess introduces the acidic polymer into an aqueous salt solutionwhich is slowly raised in supersaturation. One common method for raisingsupersaturation is to slowly introduce one of the ionic species, forexample using a modified vapor diffusion technique developed by Addadiet al. (Addadi, L. et al. [1985] Proc. Natl. Acad. Sci. USA,82:4110-4114), in which ammonium carbonate (NH₄)₂CO₃ vapor, produced bydecomposition of its powder, diffuses into a solution containing calciumchloride CaCl₂ and the acidic polymeric additive. This multistageprocess is illustrated by the formula:

Alternatively, calcium phosphate (CaP) readily forms an amorphous gelprior to crystallization, which is unlike the PILP phase (a trulyliquid-like material and has a distinct phase boundary that can be actedupon by capillary forces). In the case of bone formation, neither anamorphous solid or gel would be sufficiently fluidic to be drawn intothe nanoscopic spaces of collagen, which is presumed necessary forleaving the matrix embedded with mineral precursor and ultimatelynanocrystallites. However, as seen in the SEM and TEM images in FIGS. 2Aand 2B, CaP “droplets” were generated using a combination of polyanionicadditives (polyaspartate and polyvinyl phosphonate) as shown in theequation below.6 mM CaCl₂(aq)+200 μg/ml PVP(aq)+200 μg/ml P(D)(aq)+(NH₄)₂HPO₄(v)

CaP

Energy dispersive spectroscopy (EDS) (FIG. 2C) and x-ray diffraction(XRD) were used to confirm composition and phase of the mineral, whichwas hydroxyapatite.

Typically, the crystal products are deposited onto a thin glasscoverslip (22 mm D.) that is placed in the crystallizing solution, whichcan then be examined by polarized light microscopy (including in situexamination with ultra-long-working-distance objectives), or gold coatedfor scanning electron microscopy (SEM). The exact concentration of thereactants depends on the experiment, and is provided below.

Formation of Fibrous Crystals. The formation of fibrous calciumcarbonate was accomplished by using the PILP process to deposit CaCO₃ onglass cover slips. The cover slips were cleaned by soaking overnight ina bath of 12N H₂SO₄/Nochromix solution, followed by a distilled waterrinse, and a final rinse with ethanol. Each of the cover slips wasplaced in a Falcon polystyrene petri dish (3.5 cm D.), to which wasadded 2.7 mL of a filtered 9 mM CaCl₂ solution (CaCl₂.2H₂0, 98+% pure,SIGMA). Poly(aspartic acid) was added to a final concentration of 28μg/ml (poly-L-aspartic acid-sodium salt, M_(w) (vis)=35600, SIGMA), anddistilled water was added to the petri dishes using a micropipette tobring the final volume to 3 mL. A control dish, containing no polymericadditives, was run in parallel with each of the experiments. Allsolutions were prepared with doubly distilled water and filtered using0.2 μm GELMAN ACRODISC syringe filters. The dishes were covered withstretched parafilm, into which three holes were punched, and placed in a10 dessicator. Vapor diffusion of ammonium carbonate was accomplished byadding three small vials (5 mL) of crushed ammonium carbonate (that hadbeen covered with stretched parafilm and punched with one hole) in thedessicator with the calcium/polymer solutions. The dessicators were thenheld at room temperature (25° C.) for one week, at which time the glassslides were removed from the solution, rinsed in a small beaker of waterand then ethanol. The glass slides were then examined using an OlympusBX-60 optical microscope in transmission mode, using crossed polars andan optional first-order-red wave plate.

Mineralization of Collagen with Calcium Carbonate. Three differentcollagen substrates were mineralized with CaCO₃: bovine collagen(type-I, insoluble from bovine Achilles tendon (ALDRICH), CELLAGENsponge (ICN), which is also type-I collagen that is fabricated into 1 mmthick porous sponges, and AVITENE Ultrafoam hemostatic sponge, which isa type-I collagen sponge with 2-100 μm pores. Either 2-3 strands offibrous bovine collagen, or ⅛″× 1/16″ strips of CELLAGEN sponge, wereplaced in the crystallizing dishes and the crystallization process wasperformed as described above. The bovine collagen was mineralized usinga solution of 12 mM CaCl₂ and 200 μg/ml polyaspartic acid(poly-L-aspartic acid-sodium salt, M_(w) (vis)=8600; SIGMA), followed byvapor diffusion of the carbonate. The CELLAGEN sponge was mineralizedusing 12 mM CaCl₂ and 200 μg/ml polyacrylic acid (polyacrylicacid-sodium salt, M_(w) (vis)=5100; SIGMA). The mineralized collagen wasremoved from the solution, rinsed in a small beaker of water and thenethanol. The collagen samples were then examined using an OLYMPUS BX-60optical microscope (in transmission mode, using crossed polars and anoptional first-order-red wave plate) and subsequently Au/Pd coated andexamined with a JEOL 6400 SEM instrument. The AVITENE Ultrafoam collagenwas mineralized using the same procedure for mineralizing the CELLAGENsponges by adding micromolar amounts ranging from 10-200 μm ofpolyaspartic acid (PAA, MW 5100, ALDRICH) and a calcium chloridesolution. The mineralized AVITENE Ultrafoam collagen was crushed using aliquid N₂ mortar and pestal and put into solution. The solution wassampled and specimens placed on a copper TEM grid.

Mineralization of Collagen with Calcium Phosphate. CELLAGEN sponge(ICN), which is also a reconstituted type-I bovine collagen that isfabricated into a 1 mm thick porous sponge, was taken in strips of ⅛″×1/16″ and placed in the crystallizing dishes and the crystallizationprocess was performed as described above. The bovine collagen wasmineralized using a solution of 6 mM CaCl₂ and 200 μg/mL of bothpolyaspartic acid (poly-L-aspartic acid-sodium salt, M_(w) (vis)=8600g/mol; SIGMA) and polyvinyl phosphonic acid (polyvinyl phosphonic acid,M_(w) (vis)=20000; POLYSCIENCES), followed by vapor diffusion ofammonium phosphate dibasic (SIGMA). The mineralized collagen was removedfrom solution, rinsed in a small beaker of water and then ethanol. Thecollagen samples were then examined using an OLYMPUS BX-60 opticalmicroscope (in transmission mode, using crossed polars and an optionalfirst-order-red wave plate) and subsequently Au/Pd coated and examinedwith a JEOL 6400 SEM or JEOL 6330 FEGSEM instrument. The mineralizedCELLAGEN collagen was crushed using liquid N₂ mortar and pestle and putinto solution. The solution was samples and specimens placed on a copperTEM grid.

Acid Etch and Bleach Treatment. In order to determine the structure ofthe calcium carbonate deposited on the collagen fibers, and the extentof the mineral penetration (acid and bleach respectively), themineralized composites were exposed to either a 0.1 N HCl solution (toetch mineral) ora 10% bleach solution (to degrade collagen) for 15minutes at 23° C. After treatment, the samples were thoroughly rinsed inde-ionized water in order to avoid any contamination by the remainingacid or bleach.

Scanning Electron Micrograph (SEM) Analysis. The samples (untreated,acid etched and bleached) were dried under vacuum at 30-40° C. overnightand subsequently Au/Pd coated and examined with a JEOL 6400 SEMinstrument. In addition to the mineralized samples, a control sample ofas received CELLAGEN sponge was also coated and examined.

XRD Analysis. Three different samples, approximately 1×1 cm² wereprepared for x-ray analysis. The first sample was as received,non-mineralized CELLAGEN, the second sample was mineralized without thepresence of polymeric additive, and the third sample was mineralized for7 days at 4° C. in the presence of μM quantities of PAA. The sampleswere run on a PHILIPS XRD 2500 at 40 KV and 20 mA. Since the crystalcomposite was not expected to vary between the 5 different samplesmineralized in the presence of polymeric additive, the samplemineralized for 1 cycle was chosen for XRD analysis.

TEM Analysis. The AVITENE Ultrafoam hemostatic collagen samples wereexamined using a transmission electron microscope (TEM) in Brightfield(BF) and diffraction modes. TEM analysis. Both CELLAGEN and AVITENEUltrafoam hemostatic sponge samples were examined using a transmissionelectron microscope (TEM) in Brightfield (BF), Poor Man's dark field(PMDF), and Selected Area Diffraction (SAD) modes.

EXAMPLE 1 Mineralization of Collagen with Calcium Carbonate

The PILP mineralization of bovine Achilles tendon type-I collagen showedinteresting results. The individual fibers of the bovine collagen becamecoated or encrusted by a film of CaCO₃. In some regions on the collagenfibers, the mineral film was patchy and in the form of isolated tablets(FIGS. 3A and 3B). Because there is a lack of facets in PILP formedcrystals, energy dispersive spectroscopy was used to verify that thesefilms and tablets are composed of CaCO₃. Interestingly, the CaCO₃“tablets” appear to have preferentially deposited along the fibers in abanded pattern (FIGS. 3A & 3B). It is not clear if the PILP phase wasdeposited in those locations due to topography (such as defects or kinksalong the crimped collagen backbone), or if some more specifictemplating interaction is occurring between the PILP phase and organicmatrix.

The CELLAGEN sponge (which is reconstituted bovine collagen) provides abetter substrate onto which to deposit PILP phase because it has a flatand compact surface, as seen in the SEM photomicrograph of untreatedCELLAGEN in FIG. 4A. Upon mineralization of the sponge without polymericaddition (FIG. 4B), large calcite crystals (˜20-40 μm) grew on thesurface of the sponge. Evidently collagen is a reasonably good substratefor heterogeneous nucleation of calcite. When the sponge was mineralizedusing the PILP process, by adding polymeric additive (polyacrylic acidin this case), the mineral phase deposited as a thick patchy film on thesurface of the sponge (FIG. 4C). A higher magnification view of one ofthese patches shows that the individual collagen sub-fibers are coatedor encased by the mineral (FIG. 4D), which is why the mineral coatingappears to be so thick since it is actually a composite structure. Itwas noted that this mineral coating serves as a protective layer againstbeam damage during SEM observation. For example, FIG. 5D shows a crackthat formed during SEM analysis on the non-mineralized region of thesponge (bottom half of the figure), which was halted by the mineralizedportion (upper half of the figure). In addition, the mineral encasedfibers exhibit charging without degradation, unlike the unprotectedfibers.

In FIGS. 5A and 5B, a periodic banding pattern can be seen on themineral coatings of these fibers. In the mineralized bovine collagenshown in FIG. 5A, blebbing is apparent along the length of encapsulatingmineral towards the end of the fiber. Unlike the banding pattern inFIGS. 3A and 3B, the spacing of the banding pattern here is submicron.At the top region of the photo, it can be seen that this fiber did notfully mineralize. The individual sub-fibers of the collagen becameseparated as they densified during sample dehydration (in preparationfor SEM analysis) because the ends were restrained by the surroundingmineralized tissue. This would suggest that some amount of mineral isintercalating into the large fiber bundles and coating the individualsub-fibers.

In FIG. 5B, which is mineralized CELLAGEN sponge, the blebbing is sopronounced that disc-shaped entities appear along the whole length ofthe fibers. The relative periodicity of the blebbing (˜380 μm) seems tosuggest that it is somehow related to the periodicity of the swollencollagen fibers, yet the spacing between the bands is ˜6 times thecollagen hole-overlap periodicity (64 nm), but an order of magnitudeless than the distance between the natural crimp in collagen (˜1 μm-10μm). The periodicity is not a precise match with native collagen;however, it is likely that the reconstituted collagen does not assembleinto as precise a periodic structure as natural collagen. It isinteresting that the encasement with mineral seems to capture thisperiodic structure, suggesting that perhaps the mineral ispreferentially located in the gap zones of the collagen, which upondehydration of the sample for SEM analysis, leaves those regions thickerthan the surrounding regions containing less mineral and more organicmatrix (which dehydrate and densify to a larger extent). Notably, Bradtet al. (Bradt, J. H. et al. [1999] Chem. Mater. 11:2694-2701) havedeposited HAP on collagen in the presence of poly(aspartic acid) andfound TEM evidence that some plate-like crystals of HAP were associatedwith the gaps in the collagen fibrils. Their results are interesting butit is likely that a more potent acidic protein will be required to fullygenerate a liquid-phase precursor for the calcium phosphate system.

PILP mineralization of the AVITENE ultrafoam hemostatic sponge yieldedsimilar results to CELLEGEN sponge mineralization. Samples that weremineralized in the absence of polymeric additives showed simple calcitenucleation on the surface of the collagen (FIG. 6B). Upon addition ofmicromolar amounts of (10-200 μm) polyaspartic acid (PAA; MW 5100,ALDRICH), each individual collagen fiber appeared to be mineralized(FIGS. 6C and 6D). As with the CELLAGEN, there were sections that werepartially mineralized (FIG. 6C) and those that were fully mineralized(FIG. 6D).

EXAMPLE 2 Mineralization of Collagen with Calcium Phosphate

The results for the CaP system were very similar to the CaCO₃ system,but with some differences in the mineral texture. The XRD spectrum ofthe mineralized collagen (not shown) shows peaks consistent with HA,although some peak shifts and differences in peak intensities are noted,which seem to suggest polymer-mineral interactions are prevalent(Sivakumar, M. et al. [2002] Biomaterials, 23(15):3175-3181). Themineralized CELLAGEN was also subjected to an acid etch treatment toremove excess mineral, and showed very similar results to the CaCO₃composites. A periodic banding pattern is seen for the more protectedmineral (FIGS. 8A and 8B), and crystals have a similar disk-likemorphology. At this point, it is not clear if these are single crystals,or just thick clumps of remnant mineral phase. In some regions of thesample, the mineral remnants were less organized and the morphology wasdifferent, exhibiting a more platy shape (FIG. 8C). Without being boundby theory, it is presumed that these banding patterns are due toexclusion of impurities and shrinkage of the precursor phase duringdehydration/solidification/crystallization, because similar incrementalgrowth bands in non-templated mineral depositions have been observed.

Using a cryogenic mortar and pestal, CELLAGEN sponges mineralized withCaP PILP phase were crushed into a fine powder using liquid nitrogen.The powder was then dispersed in ethanol and sonicated for 5 minutes. Asmall drop of the solution was then pipetted onto a copper TEM grid andstained using a 1% phosphotungstic acid in 0.1M PBS (ph 7.4). The gridswere then examined using a transmission electron microscope (TEM). Asobserved in FIG. 9A, the unmineralized CELLAGEN sample (which was alsoprepared in the same manner as noted above), characteristic 67-70 nmquarter-staggered banding patterns appear in the bright field image.Since collagen is a pseudo-liquid crystalline polymer, there have beenreports in the literature in which collagen has displayed a distinctdiffraction pattern. Yet, in close agreement with the XRD diffractionpatterns of CELLAGEN, the diffraction pattern of CELLAGEN in FIG. 9Bshows a similar pattern to that of the transmitted beam. Unlike FIG. 9A,where there are only banding patterns present, in the bright field imageof mineralized CELLAGEN, FIG. 9C, there are numerous black spots withinthe collagen. These black spots (1-5 nm) infer a higher electronadsorption, most often caused by crystalline materials. While there is apossibility of these spots being a deposition of phosphotunstic acidcrystals, the larger (50-70 nm) round particles highlighted by arrowscorrelate well with SEM images of the CaP PILP phase (FIG. 2B), and thusare not believed to be a resultant of the stain. More interesting,however, are the diffraction patterns of the mineralized CELLAGEN sponge(FIG. 2D). Unlike the diffraction pattern of pure collagen (FIG. 9B),the selected area diffraction (SAD) of the mineralized fiber (FIG. 9C)shows distinct diffraction along the long axis of the fiber (indicatedby arrow).

Based on these results, it has been observed that a combination ofpolymers with phosphate and carboxylate functionality elicits the PILPprocess in CaP. With the phosphate containing polymer alone, there issome evidence of PILP phase, but it appears to be less fluidic than whena combination of the two polymers is used. Therefore, polypeptidescomprising controlled amounts of aspartic acid and serine residues (forsubsequent phosporylation) using a solid-phase peptide synthesizer(ABI-433A) can be utilized. Fortunately, relatively low molecular weight(M.W.) polymers seem to be the most effective at inducing the PILPprocess (e.g., within the range of about 50 to about 75 residues), whichmakes this synthetic route amenable for preparing small amounts of the“mimetic proteins” of interest. The following series of polypeptides canbe used for further studies, and based on the results of these studies,tailor-made polymers can be designed for further optimization: (i)homopolymer of polyserine, phosphorylated to different degrees(M.W.=5000 Da); (ii) alternating copolymer of Asp and Ser residues (withvarying degrees of phosphorylation) (M.W.=5000 Da); (iii) alternatingcopolymer of Asp, Ala, PSer, for reduced polarity (M.W.=5000 Da); andblock copolymer of Asp and Ser residues (with varying degrees ofphosphorylation) (M.W.=5000 Da), block size of about 10 residues.

The polymers can then be examined under similar conditions that wereused for the previous studies, with variable concentrations of thepolymeric additive. Based on the findings of these studies, furtheroptimization and trends can be determined using statistically designedexperiments (with other factors to consider, such as: Ca concentration,rate of phosphate additions, polymer concentration and M.W., Mg-ionadditive, carbonate additive, and temperature).

EXAMPLE 3 Crystallographic Orientation of Calcium Phosphate on CollagenFibrils

A reconstituted collagen sponge (Cellagen., ICN Biomedicals Inc.), whichis comprised of pure type-I bovine collagen, was mineralized with HAmineral using the PILP process. The process involved using a 1 mMCaCl₂.2H₂O solution in combination with 200 μg/mL Poly(αβ-DL-Asparticacid) (Polyasp) and 200 μg/mL Poly(vinyl phosphonic acid) (PVPA). Thecollagen sponge was cut into a 3×3 cm² sample and placed in themineralizing solution. This solution was then placed, uncovered, in adesiccator with three equal sized petri dishes, uncovered, and filledwith crushed diammonium hydrogen phosphate. Uncovered is stressed inthis explanation because, in the CaCO₃ PILP process, all of the dishesare covered with parafilm into which three holes are punched to reducethe diffusion rate of the reactants. The ammonium carbonate decomposesmuch more rapidly than the ammonium phosphate; therefore, the reactionkinetics are decreased. The entire desiccator was then placed in anincubator set at 37° C. The ammonium phosphate slowly decomposed intothe vapor phase and fed the phosphate counterion into the mineralizingsolution. The samples were left to mineralize between 1-4 weeks, atwhich time they were removed from solution and rinsed in both water andethanol to remove any soluble salts.

Samples were prepared for transmission electron microscopy (TEM)following the protocols performed on bone and naturally mineralizedtendon by Weiner and Traub (Weiner, S. et al., Journal of StructuralBiology, 1999, 126(3):241-255). This included crushing the samples intoa nanometer powder in a liquid nitrogen mortar and pestle. A few smalldrops of ethanol were then placed on the powder, followed by drawing theslurry into a micropipette. The slurry was transferred to a 3 mmdiameter carbon/Formvar coated copper TEM grid, followed by stainingwith 1% phosphotungstic acid (PTA) in a PBS buffer. The samples werethen analyzed using a 200cx JEOL TEM at 200 kV in brightfield (BF),weakbeam darkfield (WBF) and selected area diffraction (SAD) modes.Normal brightfield images are produced using the transmitted spot toproduce the brightest image possible. When a crystal phase is present,all of the planes that are parallel to the electron beam will diffract,thus creating diffraction patterns, such as those observed in FIG. 11B.When an objective aperture is placed over a diffraction spot(diffraction plane), the only electrons that are available for imagingare those originating from that plane. This converts a BF image into aPMDF image, in which the only crystals of the image that are illuminatedare those that created the selected diffraction spot.

Samples were also prepared for scanning electron microscopy (SEM) byallowing whole samples to dry in air, mounting the dried samples on analuminum stub covered in double-sided copper tape, and then sputtercoating the stub with either a Au/Pd or amorphous carbon film. Thesamples were then analyzed using either a 6400 JEOL SEM or a 6330 JEOLFEGSEM at 15-20 kV. Elemental x-ray analysis (EDS) was performed on themineralized sample with a Link-ISIS which was attached to both SEMs.

HA has a hexagonal crystal structure, which is best described by MillerIndices using the 4 coordinate axis, and therefore the directions arenot mutually perpendicular to planes as occurs in cubic systems(Materials Science and Engineering: An Introduction; 5^(th) Edition,William D. Callister, Jr., John Wiley & Sons, Inc., 2000, chapter 3, pp.30-65). The only directions that are mutually perpendicular are the{001} family of planes. The structure factor of the (001) plane does notallow it to appear in reciprocal space, and only the higher order (002)and (004) spacings are diffracted, but the [001] direction is alwaysreported for the growth direction of HA in bone. As for the periodlisted in the (21.1) ring, it was done to show that true hexagonalnomenclature should be reported when using the 4 coordinate system toindicate a crystallographic plane. The (002) and (004) are higher orderreflections of the basal planes of HA, and thus the third number (whichis represented by a period in the (21.1) plane) is implied.

The present inventors have shown that collagen/HA composites can becreated in which each individual collagen fibril is fully encased andinfiltrated with mineral through the PILP phase, as shown in FIG. 10A.From the SEM micrographs it is not readily apparent as to theorientation of the HA in the collagen. Using a field-emission SEM(FEGSEM), higher resolution images of mineralized collagen fibrils havebeen obtained which show the platy/needle-like crystals oriented alongthe long axis of the collagen fibril, as shown in FIG. 10B. While thehigher magnification images suggest that the mineral is aligned, it doesnot automatically provide crystallographic orientation. Usingtransmission electron microscopy (TEM), the present inventors haveproduced data showing that this mineral is indeed aligned in the [001]direction along the long axis of the collagen fibrils, similar to themineral crystals found in bone.

Using transmission electron microscopy (TEM), the present inventors havebeen able to isolate a single collagen fibril mineralized with HA PILPphase (as shown in FIG. 11A). Although the mineral is not easilyvisualized in the brightfield image, using selected area diffraction ofthe middle of the fiber yields a diffraction pattern identified as HA(as shown in FIG. 11B). More importantly, the arcs for the 002 and 004reflections are observed, meaning that the crystals are aligned parallel(c-axis of HA) to the long axis of the collagen fibril, such as isobserved for mineral in bone.

While this diffraction pattern itself is intriguing data, what makes itmost impressive is that it is identical, from the (21.1) diffractionring to the arcing of the (002) and (004) diffraction planes, tomineralized collagen in bone. FIG. 12 shows a side-by-side comparison ofdiffraction data from the mineralized collagen fibril of the presentinvention to that of natural bone. It is important to note that whileresearchers have observed collagen to diffract x-rays due to itsanisotropic orientation (yielding arc patterns typical of orientedfibrous materials), the small scale of isolated collagen fibers that isused for electron diffraction is not sufficient to diffract electrons,as is seen in FIG. 9B of an SAD pattern of collagen fibers prior tomineralization. Therefore, it can be concluded that the diffractionpattern in FIG. 11B could not be from the collagen by itself. Asmentioned, the HA mineral phase is not easily observed within thecollagen fibril. This was easily overcome using Poor Man's darkfield(PMDF), a technique that uses only the electrons diffracted fromselected planes to image a sample.

Placing the objective aperture over the (002) and then imaging usingPMDF demonstrates that long platy/needle-like crystals oriented with thelong axis of the collagen fibril (as shown in FIG. 11C). This PMDF data,combined with the extreme similarity of the diffraction patterns betweenthe sample of the invention and natural bone confirms that this isindeed the HA phase of calcium phosphate. The only remaining question iswhether or not this is actually mineralized collagen or if it is simplyHA crystals. Two pieces of evidence refute any claims to this challenge.The first piece of evidence is thatin SEM images of this exact region,platy/needle crystals cannot be discerned (as shown in FIG. 13A) butinstead, a non-descript fiber of diameter ˜150 nm with smooth surfacesis seen, indicating that the needle-like crystals observed in PMDF areintrafibrillar (EDS analysis on this bundle confirms the presence of Ca,P, and O (as shown in FIG. 13B)). The second piece of evidence is thatin the same bundle of mineralized collagen, in which the mineralizedfiber in FIGS. 11A and 11B was found, there was a section of fibers thatdisplayed a faint banded pattern that matches with the 64 nm bandingpattern of collagen (as shown in FIG. 13C), thereby confirming that theremainder of the bundle is mineralized collagen.

This data suggests that after mineralizing type-I collagen via apolymer-induced liquid-precursor (PILP) process (CaP variety), themorphology of mineralized collagen tissues in bone and teeth (at thenanostructural level) has been recreated. At early times in themineralization process, the present inventors have shown amorphous PILPdroplets in association with type-I collagen, which suggests that duringsynthesis these droplets are being drawn into the collagen via capillaryaction. At later times, as the amorphous PILP phase begins tocrystallize, the collagen-mineral interface leads to orientation of theHA crsytals in the [001] direction (along its c-axis), leaving itintrafibrillarly mineralized with the classic “deck-of-cards”architecture described for bone (Weiner & Traub). EDS analysis of thisfibril confirms the presence of Ca, P, and O within the fibril. PMDF,with the (002) HA planes highlighted, demonstrates that longplaty/needle-like crystals are oriented along the long axis of thecollagen. Finally, as the inventors' previous SEM data have shown, theexact mineralized collagen fibril analyzed for the TEM orientation datahas a non-descript appearance, meaning that the platy mineral isintrafibrillarly mineralized within the 150 nm diameter fibril.

EXAMPLE 4 Mineralization of Polymeric, Fluid-Swellable Materials

The experiments described above with respect to collagen were similarlycarried out on SCOTCH BRITE sponges.

FIG. 14 shows an SEM of a SCOTCH BRITE sponge that has not beenintroduced to mineralizing solutions. It has micropores on the order of0.75 microns-2.0 microns in diameter. FIG. 16 shows an SEM of a SCOTCHBRITE sponge that has been mineralized using the process of the subjectinvention. The sponge has been coated with non-equilibrium morphologycalcium carbonate, thus clogging some of the pores. Specifically, thesponge shown in FIG. 16 was mineralized four times (three days for eachmineralization interval at 12 mM CaCl₂, 200 μg/mL PAA (5100 m.w.), vapordiffusion of ammonium carbonate). After each interval, the solution wasrefreshed. Although the calcium carbonate coating is not obvious in theSEM of FIG. 16, energy dispersive spectroscopy (EDS) measurements havebeen performed which ensure that the sponge was mineralized with calciumcarbonate. In addition, whereas the unmineralized sponge would beextensively damaged by the electron beam from the EDS measurements (asorganic materials do not conduct electricity with great ease), themineralized samples, as the one shown in FIG. 16, resisted electron beamdamage due to the inorganic coating.

EXAMPLE 5 In Vitro Cellular Response to Mineralized CELLAGEN

Rat bone marrow stem cells were harvested and seeded into 168 cm²culture flasks in standard culture media (DMEM supplemented with 10%FBS, 1% Penicillin/Streptomycin and dextrose). Cells were cultured for14 days and media was changed on every fourth day. On day 14, the cellswere trypsinized, counted and seeded in four 168 cm2 tissue culturedishes at a density of 1E6 cells/cm². Stem cells in three culture disheswere supplemented with OS media (Standard media+100 nM Dexamethasone+10nM b-Glycerophosphate+0.05 mM Ascorbic acid) for an eight-day period toinitiate and accelerate stem cell differentiation into osteoblasts. Theremaining stem cells were supplemented with standard culture media toserve as controls.

Following this eight-day supplementation period, the osteoblasts andstem cells were trypsinized and counted with a hemocytometer.Osteoblasts and undifferentiated stem cells were seeded into three wellseach of a 24-well plate at a density of 1.6E4 cells/well. The cells wereallowed to attach to the base of each well for a 3.5-hour period atwhich time an alkaline phosphatase (ALP) assay was performed to evaluatethe extent of differentiation to osteoblast-like cells elicited by theOS media. Scaffolds composed of unmineralized CELLAGEN (A, n=6),mineralized CELLAGEN without polymer (B, n=6), and mineralized CELLAGENwith polymer (C, n=6) were sterilized using 2.5 Gy gamma irradiation.Two scaffolds per matrix type were reserved for negative control (nocells) SEM analysis; the remaining 4 scaffolds per matrix type wereseeded with cells. Osteoblasts were seeded at a density of 4E4cells/scaffold and aliquoted onto each scaffold in a total of 80 ul. Allscaffolds were placed in separate 2 cm wells of a 24 well plate.Osteoblasts were allowed to adhere to the scaffolds for a period of 3.5hours, at which time the cell-containing scaffolds were removed andplaced into new 2 cm² wells. The number of cells remaining in the 2 cm²incubation wells after removal of the scaffolds were quantified using acrystal violet assay. The cell-containing scaffolds were thensupplemented with standard media and kept in a 37° C. incubator for aseven-day period. After 12 hours and 7 days, osteoblasts were fixed inKarnovsky's solution and prepared for SEM imaging.

MSCs differentiated into the osteoblast lineage when cultured with OSmedia as confirmed with the ALP assay. After scaffold exposure to 4E+04cells/scaffold for 3.5 hours, 25% of the cells adhered to theunmineralized CELLAGEN scaffold whereas 50% of the cells adhered to themineralized CELLAGEN scaffold with or without polymer (FIG. 21).

Absorbance values were compared to a previously constructed standardcurve for determination of cell number. Initial comparisons with thestandard curve were used to indicate the number of cells in the base ofeach 2 cm² well, and represent the number that had not successfullyadhered to the scaffolds. The difference between this number and 4E+04is the total number of cells that had successfully adhered to eachmatrix. Since all scaffolds that received osteoblasts were seededsimultaneously, the numbers given above were assumed to be valid for allscaffolds even though only 2 per treatment underwent this preliminarycrystal violet stain and cell number determination.

Qualitative SEM imaging indicated adherence of osteoblasts onto themineralized matrices (FIG. 22A). The presence of calcified matrixnodules (FIG. 22B) suggests interaction of differentiated osteoblastswith the scaffold. Positive identification of osteoblast within thematrix will be confirmed in future studies by quantifiable methylthiazoltetrazolium (MTT) proliferation assays. FIG. 21 summarizes results ofcell adhesion to the mineralized CELLAGEN scaffolds. The mineralizedscaffold without polymer (containing blocky calcite crystals) showedsimilar cell adhesion.

EXAMPLE 6 Organic/Inorganic Composites Having NanostructuredArchitecture of Bone

Using a PILP phase, the process of the present invention provides ameans for generating intrafibrillar mineralization of organicsubstrates, such as collagen, yielding composites that are similar incomposition and architecture as bone. However, the hierarchical levelsof structure found in bone, which result from cellular processes, aredifficult to duplicate synthetically. Therefore, the present inventorshave developed methods to impart some of the more significant featuresof those of natural bone, such as microporosity for cell infiltration,and laminated structures for enhanced mechanical properties, to thecomposites of the present invention.

Compact bone has numerous osteonal canals. These osteons are comprisedof concentric lamella of mineralized collagen (Weiner S, Traub W, WagnerH D [1999] Journal of Structural Biology 126:241-255). As previouslyindicated, the ability to create such a complex structure in vitro byartificial mineralization processes is not possible. However, thepresent invention can overcome this hurdle. Using the process of theinvention, layers of mineralized collagen films can be individuallymineralized via the PILP mechanism. These layers, whether composed ofpre-oriented or isotropically arranged collagen fibers, can then be cutinto desired dimensions and wrapped around a mandrel 10. As shown inFIGS. 22A-22E, the mandrel 10, which represents the space occupied bythe canal in natural osteons, will be used as a mechanical support (orform) in which to wrap the mineralized (or pre-mineralized) films orsheets 20. The mandrel 10 can be composed of a bioresorbable material,in which case it can remain as part of the composite. Alternatively, themandrel 20 can be composed of a non-resorbable material, in which caseit can be removed before implantation into a patient.

Since the mineralized layers do not have any natural tacticity, acoating of bioresorbable adhesive 30 (e.g., polylactic acid,polyglycolic acid) can be applied between each individual layerwrapping. Alternatively, a cementations adhesive layer can be applied,either via the PILP process of the present invention, or other bonepaste materials, mimicking that of the “cement line” in natural bone.Once each individual osteon 50 is fabricated, many osteons 50 can beadhered to one another, as shown in FIG. 22E, to form a much largercomposite resembling the hierarchical structure of osteonal bone.

The organic fluid-swellable, fibrous matrix of the composites of thepresent invention can be made so as to be oriented to achieve, forexample, a parallel orientation, using flow fields, electric fields,magnetic fields, or combinations thereof (Murthy N. S. [1984]Biopolymers 23:1261-1267; Oh Y. R. et al. [1992] J. Chem. Eng. Jpn.25:243-250; Dickinson R. B. et al. [1994] Ann. Biomed Eng. 22:342-356;Tranguillo R. T. et al. [1996] Biomaterials 17:349-357; Guido S. et al.[1993] J. Cell. Sci. 105:317-331). During the process of the invention,such oriented substrates act as templates for deposition of theamorphous mineral precursor into the fluid-swellable matrix. Therefore,an oriented nanocrystalline hydroxyapatite phase can be achieved, forexample. Such a composite would more closely mimic the structure ofparallel-fibered bone, providing important advantages such as tailoredmechanical properties and contact guidance of cells during healing.

In one embodiment, the mineralized collagen sheets 20 can be adhered toone another by bioresorbable adhesives, such as those previouslydescribed, with each mineralized collagen layer placed in alternatingorientation, to for a cross-ply architecture, as shown in FIGS. 23A-C.

EXAMPLE 7 Porous Organic/Inorganic Composites

The ends of long bones have a much different macro-structure than thatof the central shaft area of long bones. Underlying the cortical bone istrabecular or cancellous bone, which has a porous structure (Weiner S etal. [1999] Journal of Structual Biology, 126:241-255).

This type of macro-structure can be achieved using the process of thesubject invention, in combination with certain processing techniquesknown in the art that can be applied to the organic matrix before,during, or after mineralization. For example, using a freeze-dried ordirect templating process, collagen scaffolds with desired porosity canbe produced with a range of pore sizes. Pore sizes in the range of about50 to 500 microns (preferably, 100 to 200 microns) are commonly used toenhance cell infiltration (osteoconductive material), which providesaccessibility to the cells for remodeling the synthetic substitute intonatural bone. These collagen scaffolds can then be mineralized via thePILP mechanism of the present invention in order to create a hard porouscomposite. Porous scaffolds can also be produced using organicsubstrates other than collagen.

Due to the size of the pores, larger homogenous structures can beprepared, which will enhance the capillary action of the secondaryliquid phase produced by the PILP mechanism, allowing the precursorphase to be drawn further into the interior of the composite, thusallowing for complete mineralization.

Porosity of the composite can also be introduced and/or controlled by avariety of techniques, including freeze drying techniques; incorporationof porogens (e.g., salts or other solubilizing substances that areremoved subsequent to matrix formation); phase segregation of matrixmedia comprised of multiple phases (e.g., block copolymers or immiscibleblends), e.g., using phase segregating polymers; high temperaturesintering; and supercritical processing (creation of matrix in asupercritical state, which generates pores as the supercriticalconditions are removed, most commonly as reduction in pressure ortemperature) (Nehrer, S., et al. [1999] Biomaterials, 19:2313-2328;Zhang, R. et al. [1999] J. Biomed. Mater. Res. 44:446-455).

As indicated above, in order to obtain large samples with controlledporosity, a freeze-drying technique can be used. The porosity ispreferably a continuous network to provide an open path for cells totravel to the center of the implant during healing. In addition, the useof the freeze-drying technique will result in a dense collagen scaffoldsuch that subsequent mineralization will lead primarily tointrafibrillar mineralization, without mineralization of interfibrillarvoids that would generate brittle regions of pure mineral phase. Theintrafibrillar mineralization should lead to a nanostructured compositewith enhanced mechanical properties that better mimic the properties ofnatural bone.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

1. A method for treating a bone defect comprising applying anorganic/inorganic composite to the site of the bone defect, wherein saidorganic/inorganic composite comprises a fluid-swellable, fibrous matrixand an inorganic mineral phase, and wherein said inorganic mineral phaseis absorbed into said fluid-swellable, fibrous matrix when saidinorganic mineral phase is in the form of a liquid-phase mineralprecursor, which subsequently solidifies and is aligned along the longaxis of each of the fibers of said fluid-swellable, fibrous matrix towhich said inorganic mineral phase is absorbed.
 2. The method accordingto claim 1, wherein said organic/inorganic composite is applied as aninjectable liquid, a film, a malleable putty, a malleable paste, aparticulate, or a molded or preformed solid.
 3. The method according toclaim 1, wherein said fluid-swellable, fibrous matrix comprises amaterial selected from the group consisting of collagen, elastin,chitin, chitosan, cellulose, and peptide nanofibers.
 4. The methodaccording to claim 1, wherein said inorganic mineral is selected fromthe group consisting of calcium phosphate, calcium carbonate,hydroxyapatite, strontium carbonate, and calcium sulfate, calciumoxalate, magnesium-bearing calcium carbonate, and magnesium-bearingcalcium, or combinations thereof.
 5. The method according to claim 1,wherein said organic/inorganic composite further comprises abiologically active agent.
 6. The method according to claim 1, whereinsaid organic/inorganic composite is porous.
 7. The method according toclaim 1, wherein said organic substrate is biocompatible andbioresorbable.
 8. The method according to claim 1, wherein saidfluid-swellable, fibrous matrix comprises a scaffold seeded with cells.9. The method according to claim 8, wherein said cells are selected fromthe group consisting of bone marrow stem cells, osteoblasts,osteoclasts, osteocytes, blood cells, epithelial cells, odontoblast,ameloblasts, and neural cells, or combinations thereof.
 10. The methodaccording to claim 1, wherein said fluid-swellable, fibrous matrix is afilm.
 11. The method according to claim 1, wherein saidorganic/inorganic composite comprises a plurality of saidfluid-swellable, fibrous matrices arranged as laminae.
 12. The methodaccording to claim 11, wherein said lamellae are arranged concentricallyaround a central void for passage of endogenous or exogenous cells. 13.The method according to claim 1, wherein said organic/inorganiccomposite comprises a plurality of said fluid-swellable, fibrousmatrices, and wherein said organic/inorganic composite further comprisesan adhesive layer between each of said organic substrates.
 14. Themethod according to claim 13, wherein said plurality of organicsubstrates have a parallel orientation.
 15. The method according toclaim 13, wherein said plurality of organic substrates are in analternating orientation.